The EWSR1-FLI1 family of fusion proteins results from chromosomal translocations leading to the fusion of amino-terminal sequences of low complexity (LC) from a family of RNA binding proteins (EWSR1, FUS, or TAF15) to the DNA-binding domain of an E26 transformation-specific (ETS) transcription factor (1). The EWSR1- FLI1 family of fusion proteins represents the dominant driver of the Ewing sarcoma family of tumors. Over 90% of Ewing sarcoma tumors studied to date harbor a chromosomal translocation encoding an EWSR1-FLI1 family fusion protein (2-4). The molecular mechanisms by which the EWSR1-FLI1 fusion oncoprotein transforms cells remain enigmatic. In this proposal, we will identify and interrogate the collaborating partner proteins and transcriptional effectors of the EWSR1-FLI1 fusion protein. We have developed innovative model systems in which small-molecule-regulated protein degrons (SMASh and the auxin-inducible degron) have been engineered into the endogenous EWSR1-FLI1 locus of patient-derived EWS cell lines. Our preliminary data and the proposed experiments will address three major research objectives: 1) Develop a model system for interrogation of the functional domains of the EWSR1-FLI1 fusion protein, 2) Identify protein partners that are necessary for EWSR1-FLI1 to drive oncogenesis, and 3) Define the functional effectors of the EWSR1-FLI1 oncoprotein using a pooled genetic screen. Aim 1 focuses on functional mapping of EWSR1-FLI1 in vitro and in vivo. We have used CRISPR/Cas9 to engineer auxin inducible degron (AID) and SMASh tags into the endogenous EWSR1- FLI locus in patient-derived EWS cell lines. These systems allow us to conditionally deplete EWSR1-FLI in vivo and in vitro. Aim 2 centers on the identification and characterization of endogenous protein partners of EWSR1- FLI1 using affinity proteomics. Our working hypothesis is that the tumor promoting function of EWSR1-FLI1 depends on interaction with other proteins. We used CRISPR/Cas9 to engineer an epitope tag into the endogenous EWSR1-FLI1 locus of EWS cells. Using this system, we will capture and characterize endogenous EWSR1-FLI1 protein complexes. Aim 3 is a functional genomic identification of EWSR-FLI1 functional effectors by CRISPR -inhibition and -activation screening. We will couple EWSR1-FLI1 depletion to CRISPR-inhibition and -activation screens to identify the functional effectors of the EWSR1-FLI1 oncoprotein.